Physical Enrichment of Transposon Mutants from Saturation Mutant Libraries Using the TraDISort Approach

Transposon-insertion sequencing methods are finding their way into the molecular toolbox of many fields of microbiology. These methods can identify the genomic locations and density of transposon insertions in saturated transposon mutant libraries and can be used to make inferences on gene function. For example, where no insertions or very few insertions are identified within a gene in a mutant library grown under permissive conditions, the gene may be essential. Furthermore, where mutations are enriched or lost in a gene after passaging the library through a selective process, the gene is likely to be involved in phenotypes linked to the process. Typically, a fitness based selection such as a stress condition is used in these experiments and the processed sequencing data are used to identify genes required for fitness under the selection. Our research team recently expanded the utility of the transposon directed insertion sequencing (TraDIS) method by applying a physical separation of a transposon mutant library mediated by fluorescence activated cell sorting, rather than a fitness-based selection. This approach, which we have named "TraDISort" is significant because it allows the study of phenotypes that are not linked to cell survival. The TraDISort approach has a broad range of future applications, in drug development, metabolic engineering and in studies of basic bacterial cell physiology.